Cross-linking of low and high molecular weight polysaccharides preparation of injectable monophase hydrogels and polysaccharides and dydrogels thus obtained

ABSTRACT

A process for the crosslinking of at least one polymer selected from polysaccharides and derivatives thereof, which is carried out in an aqueous solvent by the action of an effective and non-excessive amount of at least one crosslinking agent, characterized in that it is carried out on a mixture containing at least one low-molecular weight polymer and at least one high-molecular weight polymer. A process for the preparation of an injectable monophase hydrogel of at least one crosslinked polymer selected from polysaccharides and derivatives thereof is also disclosed. Crosslinked polymers and injectable monophase hydrogels, respectively, are obtainable by each of said processes.

The present invention relates to:

a novel process for the crosslinking of at least one polymer selectedfrom polysaccharides and derivatives thereof;

a process for the preparation of an injectable monophase hydrogel of atleast one such polymer; and

the crosslinked polymers and injectable monophase hydrogels respectivelyobtainable by each of said processes.

The hydrogels in question, based on said crosslinked polymers, havenumerous outlets, especially as filling materials in plastic, cosmeticand dental surgery, in ophthalmology, in orthopedics, etc., as productsfor preventing tissue adhesions, in general surgery, in urology, etc.Said hydrogels are particularly suitable for repairing vocal cords. Theoutlets indicated above for products of this type, without implying anylimitation, are familiar to those skilled in the art.

The invention is the result of a genuine effort to optimize theoperation of crosslinking the polymers in question with a view toobtaining injectable monophase hydrogels that are of particular value inrespect of the following compromise: on the one hand mechanicalproperties and remanence, and on the other hand injectability (withacceptable injection forces and injection needle diameters).

It is pointed out here that the term “injectable” employed in thepresent text, with reference to both the hydrogels of the prior art andthe hydrogels of the invention, denotes manual injectability by means ofsyringes equipped with conventional needles (having a diameter ofbetween 0.1 and 0.5 mm). Within the framework of the present invention,it is possible in particular to formulate hydrogels that can be injectedthrough hypodermic needles of 30 G½, 27 G½, 26 G½ and 25 G.

According to the prior art, hydrogels, especially injectable hydrogels,have already been prepared from polysaccharides and derivativesthereof—especially hyaluronic acid salts—having a zero, low or highdegree of crosslinking.

With reference to the specific problem of injectability, biphasecompositions have been proposed whose continuous phase, in particular,is based on such hydrogels. The continuous phase serves as aplasticizer, injection vehicle for a disperse phase. This disperse phaseis more or less solid and more or less differentiated from thecontinuous phase. Thus:

the biphase compositions described in patent application EP-A-0 466 300consist of two bioabsorbable phases—continuous and disperse—and take theform of slurries. Said two phases are advantageously prepared fromfibers of Hylan (natural hyaluronic acid chemically modified in situ inorder to facilitate its extraction from the tissues);

the biphase compositions described in patent application WO-A-96 337 51also have two bioabsorbable phases with a better separation, thedisperse phase consisting of insoluble fragments of a highly crosslinkedpolymer hydrogel (selected from hyaluronic acid and its salts);

the biphase compositions described in patent application WO-A-00 014 28contain a non-bioabsorbable disperse phase (particles of at least onehydrogel of a (co)polymer obtained by the polymerization andcrosslinking of acrylic acid and/or methacrylic acid and/or at least onederivative of said acids) suspended in an aqueous solution of acrosslinked or non-crosslinked polymer selected from proteins,polysaccharides and derivatives thereof.

These biphase systems are not fully satisfactory insofar as they areassociated with justifiable fears of uneven flow during injection andparticularly after injection, a more rapid disappearance of thecontinuous phase (having a zero or low degree of crosslinking) and hencean at least partial loss of the desired effect, especially fillingeffect.

Monophase hydrogels, developed from the same types of polymers, weretherefore also proposed in parallel.

In patent applications WO-A-98 356 39 and WO-A-98 356 40, the product inquestion is not an injectable hydrogel but a product of solidconsistency. Said patent applications in fact describe ocular implantsused to temporarily fill a surgically created void. The hydrogeldeveloped in U.S. Pat. No. 4,716,154 is proposed as a substitute for thevitreous body. The polymer in question (sodium hyaluronate) has a verylow degree of crosslinking in order to obtain an injectable hydrogel.The monophase hydrogel described in patent application WO-A-02 057 53 isladen with an antiseptic that is effective in protecting it from freeradicals after implantation. Patent application WO-A-02 063 50 describesa process capable of generating this type of hydrogel that is veryhomogeneous throughout.

All these monophase hydrogels were obtained from high-molecular weightpolymers crosslinked using an effective and non-excessive amount of atleast one crosslinking agent, in an aqueous solvent.

In the light of this prior art, the inventors wished to improve theefficacy of crosslinking of the polymer in question, especially in orderto improve the degradation resistance (remanence) of the implantedhydrogel while at the same time preserving the possibility of injectingsaid hydrogel under acceptable conditions.

To improve the crosslinking efficacy, the inventors initially consideredusing more crosslinking agent. This approach was quickly discarded onthe grounds that it inescapably causes denaturation of the polymer inquestion and chemical contamination of the crosslinked product obtained.

Said inventors then considered increasing the concentration of polymerin the reaction mixture. In the same way, this second approach had to bediscarded, a priori, because of the polymers conventionally usedhitherto, namely high-molecular weight polymers. Thus sodium hyaluronateis always used with high molecular weights (Mw>10⁶ Da, ≈2.10⁶ Da, 3.10⁶Da) at concentrations close to the maximum concentration, which is about105-110 mg/g. Using it at a higher concentration is difficult (theviscosity of the reaction mixture becomes too high) and inescapablycauses problems of solubility, poor homogeneity, etc.

Concentrating the reaction medium, on the other hand, is found to bepossible with low-molecular weight polymers (sodium hyaluronate ofmolecular weight 300,000 Da, having an intrinsic. viscosity of 600 ml/g(those skilled in the art are perfectly familiar with the relationshipbetween these two parameters: molecular weight (M) and intrinsicviscosity (η), which is given by the Mark-Houwink formula: M=kη^(α), thevalues of k and α depending on the nature of the polymer in question),can be concentrated from 110 to 200 mg/g). Unfortunately the crosslinkedpolymer obtained generates an inhomogeneous, injectable biphase hydrogelunder these conditions.

In such a context, the inventors surprisingly established thatassociating low-molecular weight polymer(s) with high-molecular weightpolymer(s) affords an excellent compromise, namely the possibility ofgenerating, for a non-excessive degree of crosslinking (equivalent tothat of the prior art), an injectable monophase hydrogel which hasimproved mechanical and remanence properties. This low-molecularweight/high-molecular weight association makes it possible to obtain ahydrogel that more than satisfies the following specifications:

monophase;

better mechanical properties and remanence than the equivalent productsof the prior art;

unaffected or even improved injectability that is still possible withconventional injection forces using conventional injection devices.

The key factor of the crosslinking process of the invention thereforelies in the concentration of the reactants (which is greater than thatof the reaction mixtures of the prior art due to the use oflow-molecular weight polymer(s)), although the crosslinking of saidconcentrated reactants is “governed” by the use of high-molecular weightpolymer(s), which guarantee the homogeneity of the crosslinked productobtained and then of the hydrogel obtained.

According to its first subject, the present invention therefore relatesto a process for the crosslinking of at least one polymer selected frompolysaccharides and derivatives thereof, which is carried out in anaqueous solvent by the action of an effective and non-excessive amountof at least one crosslinking agent, said process being improved in thatit is carried out on a mixture containing at least one low-molecularweight polymer and at least one high-molecular weight polymer.

Said mixture of course contains said low-molecular weight polymer(s) ina sufficient amount to guarantee a relatively high concentration ofpolymer(s) in the reaction medium, and said high-molecular weightpolymer(s) in a sufficient amount to guarantee that said crosslinkedpolymer obtained has a homogeneous consistency.

The crosslinking process of the invention is a process for thecrosslinking of polymers selected from polysaccharides and derivativesthereof. The polymer(s) in question can therefore be natural orsynthetic. Examples of natural polymers are hyaluronic acid and itssalts, other glycosaminoglycans such as chondroitin sulfates, keratansulfate, heparin and heparan sulfate, alginic acid and its biologicallyacceptable salts, starch, amylose, dextran, xanthan, pullulan, etc.Examples of synthetic derivatives of natural polysaccharides are carboxycellulose, carboxymethyl cellulose, alkyl celluloses such ashydroxyethyl cellulose and hydroxypropyl methyl cellulose (HPMC),oxidized starch, etc.

The process of the invention is suitable for the crosslinking of any oneof these polymers insofar as said polymer is used with low and highmolecular weights.

The process of the invention is suitable for the crosslinking ofmixtures of such polymers, said mixtures containing at least onelow-molecular weight polymer and at least one high-molecular weightpolymer.

The terms “low” and “high” applied to the molecular weights in questionobviously cannot be defined more precisely at this stage of thedescription of the invention since they depend on the mixture inquestion and the nature of the polymer(s) present. Likewise, it is notgenerally possible to indicate the relative proportions in which thepolymer(s) present is(are) used. However, those skilled in the art havea perfect understanding of the spirit of the invention, which is toconcentrate the reaction medium containing the low-molecular weightpolymer(s), but to introduce at least one high-molecular weight polymerto moderate and control the crosslinking in question. The aim is toobtain a coherent crosslinked product that is the precursor of amonophase hydrogel. It is desirable to avoid the formation of lumps thatmay be coherent when crosslinking has ended, but capable of losing theircoherence when the injectable hydrogel is prepared.

The above explanations are given a posteriori. The result obtained wasin no way predictable.

Within the framework of one advantageous variant, the reaction mediumcontains a single polymer which is used with at least two differentiatedmolecular weights, at least one being low and at least one being high.Within the framework of this advantageous variant, the same polymer ispreferably used with a single low molecular weight and a single highmolecular weight.

The polymer in question is advantageously a hyaluronic acid salt. It isvery advantageously selected from the sodium salt, the potassium saltand mixtures thereof. It preferably consists of the sodium salt (NaHA).

In the context of the crosslinking of this type of polymer, thoseskilled in the art understand that said crosslinking is carried out in abasic aqueous solvent. In general, said crosslinking is obviouslycarried out under pH conditions that favor the dissolution of thepolymer in question.

In the context of the crosslinking of this type of polymer (hyaluronicacid salt(s)), in one preferred variant of carrying out thecrosslinking, the reaction mixture contains:

at least one hyaluronic acid salt of low molecular weight m, wherem≦9.9.10⁵ Da, advantageously 10⁴ Da≦m≦9.9.10⁵ Da; and

at least one hyaluronic acid salt of high molecular weight M, whereM≧10⁶ Da, advantageously 10⁶ Da≦M≦10⁸ Da and very advantageously 1.1.10⁶Da≦M≦5.10⁶ Da,

said low-molecular weight and high-molecular weight salts advantageouslybeing of the same nature and very advantageously consisting of sodiumhyaluronate (NaHA).

In such a context, said reaction mixture advantageously has an intrinsicviscosity of less than 1900 ml/g, i.e. Σω_(i)[η_(i)]₀≦1900 ml/g, whereω_(i) is the mass fraction of polymer fraction i, having an intrinsicviscosity [η_(i)]₀, in the reaction mixture. Those skilled in the artare familiar with the intrinsic viscosity parameter and are aware of thelaws of additivity of said parameter.

The condition stated above makes it possible to obtain a monophasehydrogel that is optimized in respect of its remanence and injectabilityproperties. It fixes the relative proportions of the salts of lowmolecular weight (m) and high molecular weight (M).

In the context referred to here (NaHA of molecular weights m and M), thereaction mixture advantageously contains more than 50% by weight, veryadvantageously more than 70% by weight, of at least one hyaluronic acidsalt of low molecular weight m, and hence, logically, advantageouslyless than 50% by weight, very advantageously less than 30% by weight, ofat least one hyaluronic acid salt of high molecular weight M.

In general, to obtain the expected effect, there is at least 5% byweight of at least one hyaluronic acid salt of high molecular weight Min the reaction mixture.

The crosslinking process of the invention is advantageously carried outwith the sodium salt of hyaluronic acid used with one low molecularweight m and one high molecular weight M, said parameters then veryadvantageously being as follows: m≈3.10⁵ Da and M≈3.10⁶ Da.

Any agent known for crosslinking polysaccharides and derivatives thereofvia its hydroxyl groups can be used as the crosslinking agent with alltypes of polymer, said crosslinking agent being at least bifunctional inorder to ensure crosslinking, an epoxy compound or derivatives thereofbeing used in particular.

It is recommended to use bifunctional crosslinking agents, by themselvesor in a mixture. It is particularly recommended to use epichlorohydrin,divinyl sulfone, 1,4-bis(2,3-epoxypropoxy)butane (or1,4-bisglycidoxybutane or 1,4-butanediol diglycidyl ether (BDDE)),1,2-bis(2,3-epoxypropoxy)ethylene,1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and aldehydes such asformaldehyde, glutaraldehyde and crotonaldehyde, taken by themselves orin a mixture. It is very particularly recommended to use1,4-bis(2,3-epoxypropoxy)butane (BDDE).

Those skilled in the art will know how to determine the effective andnon-excessive amount of crosslinking agent(s) to use. It is recommendedto use an effective and non-excessive amount such that the degree ofcrosslinking (τ), defined by the following ratio:${\tau = {\frac{\begin{matrix}{{Total}\quad{number}\quad{of}\quad{reactive}\quad{groups}} \\{{in}\quad{said}\quad{crosslinking}\quad{agent}}\end{matrix}}{\begin{matrix}{{Total}\quad{number}\quad{of}\quad{disaccharide}} \\{{units}\quad{in}\quad{the}\quad{polymer}\quad{molecules}}\end{matrix}} \times 100}},$is theoretically between 0.5 and 70%, advantageously between 4 and 50%.

The crosslinking process of the invention is novel by virtue of theforms in which the polymers in question are used. In other respects itis carried out in conventional manner with at least one crosslinkingagent. It is noted that said crosslinking agent is generally reactedwith the dissolved polymer(s), but reacting it with said polymer(s)during hydration, by the process described in WO-A-02 06 350, is in noway ruled out.

The crosslinked product obtained after carrying out the crosslinkingprocess of the invention is generally formulated for generating thedesired injectable monophase hydrogel. If necessary, it is neutralizedbeforehand. It has been seen that the hyaluronic acid salts are actuallycrosslinked in a basic medium. The formulation is carried out in asolution buffered to a pH compatible with the human body (since thehydrogel in question is generally intended for injection into the humanbody), said pH being between 6.5 and 7.5, advantageously between 7 and7.4 and very advantageously between 7.1 and 7.3. The crosslinked polymeris in equilibrium in said solution. It also acquires an osmolaritycompatible with that of the human body. Surprisingly, after thisformulation step, the diluted crosslinked polymers of the invention aremonophase hydrogels.

In one preferred variant of carrying out the invention, an injectablehydrogel of the invention is prepared by crosslinking a mixture of atleast one polymer consisting of hyaluronic acid salt(s) (see above),neutralizing the crosslinked product obtained, and then formulating itinto a solution buffered to a pH of between 7.1 and 7.3, at aconcentration of between 10 and 40 mg/g, advantageously of between 20and 30 mg/g.

The process for the preparation of the injectable monophase hydrogelfrom the crosslinked polymer (obtained by the crosslinking processconstituting the first subject of the present invention) constitutes thesecond subject of the present invention.

We now come to the third and fourth subjects, which respectively consistof the crosslinked polymer obtainable after carrying out thecrosslinking process (first subject), and the injectable monophasehydrogel obtainable by the formulation (second subject) of saidcrosslinked polymer, as stated above.

Said polymer and hydrogel advantageously contain low-molecular weightsodium hyaluronate and high-molecular weight sodium hyaluronate, theproportion of said low-molecular weight sodium hyaluronate veryadvantageously being more than 50% by weight.

The structure of the injectable monophase hydrogel—fourth subject of thepresent invention—is novel. Its consistency is resistant to degradation.This resistance of the hydrogel is far greater than that of theequivalent products of the prior art.

Those skilled in the art are aware that one of the methods of estimatingthe consistency of a hydrogel, especially of this type, is to measurethe following parameter:${\tan \cdot {delta}} = {\frac{G^{''}}{G^{\prime}} = {{f\left( {{stressing}\quad{frequency}} \right)}.}}$

The hydrogels of the invention have the outlets indicated in theintroduction of the present text. They are found to be particularlyefficient for these purposes.

It is now proposed to illustrate the invention in its various featuresby means of the Examples below. More precisely:

Example 1 illustrates the prior art (crosslinking of a polymer of highmolecular weight);

Example 2 illustrates the remarks made in the introduction of thepresent text (crosslinking of the same polymer of low molecular weight);and

Examples 3 and 4 illustrate the invention (crosslinking of the samepolymer of low and high molecular weight, used in different relativeamounts).

These are preceded by a description of a few methods of measurement usedto characterize the products in question.

Measurement of the Intrinsic Viscosity

The intrinsic viscosity of sodium hyaluronate (NaHA) (in ml/g) isdetermined according to the European Pharmacopeia for NaHA (2.2.9) usinga capillary viscometer of the Ubbelohde type.

Measurement of the Ejection Force (No Specific Standard for This Test)

The injectability of the gel based on NaHA is determined by measuringthe force (in Newtons, N) required to eject the gel contained in astandard syringe, through a needle of 27 G½, at a rate of 12.5 mm/min.The tests were performed on a Verstatet® tensile device marketed byMecmesin.

Measurement of the Remanence

The consistency of the gel is characterized at 25° C. by rheologicalmeasurement of the moduli of elasticity (G′) and viscosity (G″) as afunction of the frequency (from 0.05 to 10 Hz), in the constantdeformation domains, using a controlled stress rheometer (Carrimed CSL500 from TA Instruments) and a cone-and-plate geometry of 4 cm 2°. Thisrheometer is checked and calibrated regularly. Degradation of thecrosslinked gel results in a change in its consistency, which ismeasured by the increase in the parameter tangent delta (tan.delta=G″/G′) as a function of time, at a frequency of 1 Hz. The gelsare degraded by being heated to a temperature of 93° C. The time afterwhich tan .delta reaches a value of 0.65 (corresponding to a degradedgel state) is measured at this temperature. A remanence index of 1(corresponding to said time) was arbitrarily set for the gel ofExample 1. The remanence index values indicated for the other gels arerelative values.

Appearance of the Hydrogel

Monophase

-   Microscopic appearance: no apparent liquid phase—fine fragmentation    of the gel into facets-   Macroscopic appearance: soft and free-flowing    Biphase-   Microscopic appearance: gel fragments bathed in a low-viscosity    liquid medium-   Macroscopic appearance: “purée” that fragments very easily—no    cohesion of the gel and no free-flowing appearance

EXAMPLE 1 High-Molecular Weight Fibers

3.5 g of sodium hyaluronate (NaHA) fibers of intrinsic viscosity 2800ml/g and moisture content 8.7% are weighed out and 25.6 g of 0.25 N NaOHare added. Hydration of the fibers takes 2 h with regular manualhomogenization using a spatula. 0.96 g of a solution of 1,4-butanedioldiglycidyl ether (BDDE) diluted to ⅕ in 0.25 N sodium hydroxide solutionis added to the reaction medium, this being followed by mechanicalhomogenization for 15 min before immersion in a thermostaticallycontrolled bath at 50° C.±1° C.

-   R=[BDDE]₀/[NaHA]₀=6%; [NaHA]_(i)=10⁵ mg/g

The reaction takes 2 h. The crosslinked product is neutralized to pH 7.2in a phosphate buffer solution and then dialyzed. The concentration ofthe resulting hydrogel is then adjusted ([NaHA]_(f)=26 mg/g) and thehydrogel is mechanically homogenized before being packed into syringesand sterilized in an autoclave by means of moist heat.

-   Injection force after sterilization: 25 N-   Remanence index of the hydrogel: 1.0-   Monophase hydrogel

EXAMPLE 2 Low-Molecular Weight Fibers

1.56 g of sodium hyaluronate (NaHA) fibers of intrinsic viscosity 600ml/g and moisture content 5.5% are weighed out and 7.15 g of 0.25 N NaOHare added. Hydration of the fibers takes 2 h with regular manualhomogenization using a spatula. 0.31 g of a solution of 1,4-butanedioldiglycidyl ether (BDDE) diluted to ⅕ in 0.25 N sodium hydroxide solutionis added to the reaction medium, this being followed by mechanicalhomogenization for 15 min before immersion in a thermostaticallycontrolled bath at 50° C.±1° C.

-   R=[BDDE]₀/[NaHA]₀=6.8%; [NaHA]_(i)=174 mg/g

The reaction takes 2 h. The crosslinked product is neutralized to pH 7.2in a phosphate solution and then dialyzed. The concentration of theresulting hydrogel is then adjusted ([NaHA]_(f)=26 mg/g) and thehydrogel is mechanically homogenized before being packed into syringesand sterilized in an autoclave.

-   Injection force after sterilization: 24 N-   Remanence index of the hydrogel: 6.0-   Biphase hydrogel

EXAMPLE 3 Mixture of Fibers

0.763 g of sodium hyaluronate (NaHA) fibers of intrinsic viscosity 600ml/g and moisture content 5.5% and 0.237 g of sodium hyaluronate fibersof intrinsic viscosity 2800 ml/g and moisture content 9.3% are weighedout. Proportions by weight in the mixture: 600/2800:77/23 (w/w).

The procedure remains identical to that of Example 2.

-   R=[BDDE]₀/[NaHA]₀=7%; [NaHA]_(i)=140 mg/g; [NaHA]_(f)=26 mg/g-   Injection force after sterilization: 15 N-   Remanence index of the hydrogel: 3.6-   Monophase hydrogel

EXAMPLE 4 Mixture of Fibers

The experiment of Example 3 is repeated, modifying the proportions byweight. Proportions by weight in the mixture: 600/2800:90/10 (w/w).

The procedure is identical to that of Example 2.

-   R=[BDDE]₀/[NaHA]₀=6.5%; [NaHA]_(i)=140 mg/g; [NaHA]_(f)=26 mg/g-   Injection force after sterilization: 14 N-   Remanence index of the hydrogel: 7.7-   Monophase hydrogel

Said Examples are summarized in the Table below. TABLE [NaHA]₀ =concentration of NaHA in the reaction medium at t₀ [NaHA]_(f) =concentration of NaHA in the final hydrogel after reaction and dilutionwith a sufficient amount of phosphate buffer G′: modulus of elasticityof the final hydrogel (Pa · s) Carrimed CSL 500 rheometer G″: modulus ofviscosity of the final hydrogel (Pa · s) Tan · delta = G″/G′ η_(int.):intrinsic viscosity of the NaHA fiber/Ubbelohde viscometer F: ejectionforce of the gel in N through a 27 G½ needle/100 N dynamometer η_(int.)(ml/g) [NaHA]_(f) in G′, G″, % = proportion by R = [NaHA]₀ final gel tan· delta F_(ap ster) Remanence n° weight in mixture m_(BDDE)/m_(NaHA)mg/g mg/g Appearance* (1 Hz) 27 G½ index 1 (100%) 2800   6% 105 26 M143/65/0.40 25 1 2 (100%) 600  6.8% 174 26 B 1300/100/0.08 24 6 3 (77%)600 + (23%) 2800   7 140 26 M 262/27/0.10 15 3.6 4 (90%) 600 + (10%)2800 6.5 140 26 M 571/41/0.07 14 7.7*M = monophaseB = biphase

The attached FIGURE shows the following curve:Tan .delta=f (stressing frequency)for each of the four hydrogels prepared according to Examples 1 to 4.

The rheological behavior of the hydrogels of the invention (Examples 3and 4) is different from that of the hydrogel of the prior art (Example1).

Furthermore, the hydrogels of the invention are monophase and thus verydifferent from the hydrogel of Example 2 (biphase).

1. Process for the crosslinking of at least one polymer selected frompolysaccharides and derivatives thereof, which is carried out in anaqueous solvent by the action of an effective and non-excessive amountof at least one crosslinking agent, characterized in that it is carriedout on a mixture containing at least one low-molecular weight polymerand at least one high-molecular weight polymer.
 2. Process according toclaim 1, characterized in that said mixture contains a single polymerwith at least two different molecular weights, at least one being lowand at least one being high, and advantageously with two differentmolecular weights, one low and one high.
 3. Process according to claim 1or 2, characterized in that said polymer is a hyaluronic acid salt. 4.Process according to claim 3, characterized in that said hyaluronic acidsalt is selected from the sodium salt, the potassium salt and mixturesthereof, and advantageously consists of the sodium salt.
 5. Processaccording to any one of claims 1 to 4, characterized in that saidmixture contains: at least one hyaluronic acid salt of low molecularweight m, where m≦9.9.10⁵ Da, advantageously 10⁴ Da≦m≦9.9.10⁵ Da; and atleast one hyaluronic acid salt of high molecular weight M, where M≧10⁶Da, advantageously 10⁶ Da≦M≦10⁸ Da, and very advantageously 1.1.10⁶Da≦M≦5.10⁶ Da, said low-molecular weight and high-molecular weight saltsadvantageously being of the same nature and very advantageouslyconsisting of sodium hyaluronate.
 6. Process according to claim 5,characterized in that said mixture has an intrinsic viscosity of lessthan 1900 ml/g.
 7. Process according to claim 5 or 6, characterized inthat said mixture contains more than 50% by weight, advantageously morethan 70% by weight, of at least one hyaluronic acid salt of lowmolecular weight m, and less than 50% by weight, advantageously lessthan 30% by weight, of at least one hyaluronic acid salt of highmolecular weight M.
 8. Process according to any one of claims 5 to 7,characterized in that said mixture contains at least 5% by weight of atleast one high-molecular weight hyaluronic acid salt.
 9. Processaccording to any one of claims 5 to 8, characterized in that saidmixture contains about 90% by weight of the sodium salt of hyaluronicacid having a molecular weight of about 3.10⁵ Da, and about 10% byweight of the sodium salt of hyaluronic acid having a molecular weightof about 3.10⁶ Da.
 10. Process according to any one of claims 1 to 9,characterized in that said crosslinking agent is selected frombifunctional crosslinking agents and mixtures thereof, is advantageouslyselected from epichlorohydrin, divinyl sulfone,1,4-bis(2,3-epoxypropoxy)butane, 1,2-bis(2,3-epoxypropoxy) ethylene,1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, aldehydes such asformaldehyde, glutaraldehyde and crotonaldehyde, and mixtures thereof,and very advantageously consists of 1,4-bis(2,3-epoxypropoxy)butane. 11.Process according to any one of claims 1 to 10, characterized in thatsaid effective and non-excessive amount of at least one crosslinkingagent is such that the degree of crosslinking, defined by the ratio:100×(total number of reactive groups in said crosslinking agent/totalnumber of disaccharide units in the polymer molecules present), istheoretically between 0.5 and 70%, advantageously between 4 and 50%. 12.Process for the preparation of an injectable monophase hydrogel of atleast one crosslinked polymer selected from polysaccharides andderivatives thereof, characterized in that it comprises: thecrosslinking of a mixture according to any one of claims 1 to 11; andthe formulation of said crosslinked mixture, neutralized if necessary,into a solution buffered to a pH of between 6.5 and 7.5, advantageouslyof between 7 and 7.4 and very advantageously of between 7.1 and 7.3. 13.Process according to claim 12, characterized in that it comprises: thecrosslinking of a mixture according to any one of claims 3 to 11; andthe formulation of said crosslinked mixture, neutralized, into asolution buffered to a pH of between 7.1 and 7.3, at a concentration ofbetween 10 and 40 mg/g, advantageously of between 20 and 30 mg/g. 14.Crosslinked polymer obtainable after a crosslinking process according toany one of claims 1 to 11 has been carried out.
 15. Injectable monophasehydrogel obtainable after a preparative process according to claim 12 or13 has been carried out.
 16. Injectable monophase hydrogel according toclaim 15, containing low-molecular weight sodium hyaluronate andhigh-molecular weight sodium hyaluronate in crosslinked form.